Gene transfer methodologies have created the opportunity for developing gene therapy for human neurological diseases such as Parkinson's Disease (PD). Adeno-associated virus (AAV) and lentivirus vectors, both of which integrate into the host cell chromosome, have been shown to provide long-term expression of genes and therapeutic efficacy in animal models of PD. These vectors, however, exhibit a number of disadvantages, among them an inability to harbor large transgene segments. Gene therapy vectors based upon the Herpes Simplex virus (HSV) offer numerous advantages for the development of novel therapeutics for PD. These include broad cellular tropism, large DNA packaging capacity that allows for expression of multiple genes, and high transduction efficiency. We propose to modify HSV-derived amplicon vectors to provide stable nigrostriatal cell-specific gene expression. For this pipeline project, we will initially construct novel integrating forms of the amplicon that will direct expression of a reporter gene product specifically within cells of the striatum and substantia nigra. DNA insulator elements will be introduced to facilitate maintenance of the integrated transcription unit in a euchromatic state. These new vectors will be co-administered with an amplicon expressing the Sleeping Beauty transposase into the striata of unlesioned rats, where gene expression duration and cell-type specificity will be experimentally derived. The vectors will then be used to deliver glial cell line-derived neurotrophic factor (GDNF) prior to 6-OHDA-induced lesioning of rats to assess the capacity of the new vector system to confer protection to the nigrostriatal pathway. The proposed studies will yield a novel HSV vector system, provide a detailed understanding of transgene expression in vivo, and evaluate its therapeutic effectiveness in protecting nigrostriatal neurons in a well-established rodent model of PD.